The dynamic association and dissociation between proteins are the basis of cellular signal transduction. This process becomes much more complicated if one or both interaction partners are intrinsically disordered because intrinsically disordered proteins can undergo disorder-to-order transitions upon binding to their partners. p53, a transcription factor with disordered regions, plays significant roles in many cellular signaling pathways. It is critical to understand the binding/unbinding mechanism involving these disordered regions of p53 at the residue level to reveal how p53 performs its biological functions. Here, we studied the dissociation process of the intrinsically disordered N-terminal transactivation domain 2 (TAD2) of p53 and the transcriptional adaptor zinc-binding 2 (Taz2) domain of transcriptional coactivator p300 using a combination of classical molecular dynamics, steered molecular dynamics, self-organizing maps, and time-resolved force distribution analysis (TRFDA). We observed two different dissociation pathways with different probabilities. One dissociation pathway starts from the TAD2 N-terminus and propagates to the α-helix and finally the C-terminus. The other dissociation pathway is in the opposite order. Subsequent TRFDA results reveal that key residues in TAD2 play critical roles. Besides the residues in agreement with previous experimental results, we also highlighted some other residues that play important roles in the disassociation process. In the dissociation process, non-native interactions were formed to partially compensate for the energy loss due to the breaking of surrounding native interactions. Moreover, our statistical analysis results of other experimentally determined complex structures involving either Taz2 or TAD2 suggest that the binding of the Taz2-TAD2 complex is mainly governed by the binding site of Taz2, which includes three main binding regions. Therefore, the complexes involving Taz2 may follow similar binding/unbinding behaviors, which could be studied together to generate common principles.

The canonical autophagy pathway in mammalian cells sequesters diverse cytoplasmic cargo within the double membrane autophagosomes that eventually convert into degradative compartments via fusion with endolysosomal intermediates. Here, we report that autophagosomal membranes show permeability in cells lacking principal ATG8 proteins (mATG8s) and are unable to mature into autolysosomes. Using a combination of methods including a novel in vitro assay to measure membrane sealing, we uncovered a previously unappreciated function of mATG8s to maintain autophagosomal membranes in a sealed state. The mATG8 proteins GABARAP and LC3A bind to key ESCRT-I components contributing, along with other ESCRTs, to the integrity and imperviousness of autophagic membranes. Autophagic organelles in cells lacking mATG8s are permeant, are arrested as amphisomes, and do not progress to functional autolysosomes. Thus, autophagosomal organelles need to be maintained in a sealed state in order to become lytic autolysosomes.

The unfolded state of proteins presents many challenges to elucidate the structural basis for biological function. This state is characterized by a large degree of structural heterogeneity which makes it difficult to generate structural models. However, recent experiments into the initial folding events of the 104-residue ribonuclease homologue onconase (ONC) were able to identify the regions in the protein that participate in the initial folding of this protein. Therefore, to gain additional structural insight into the unfolded state of proteins, this study utilized molecular dynamics simulations using the UNited-RESidue (UNRES) force field to evaluate whether there is a good agreement between the experimentally determined initial structures and the structures identified by computer simulations along a folding pathway. Indeed, these UNRES simulations accurately identified the two regions experimentally observed to form the initial native structure along the folding pathway of ONC. In addition, these regions are determined to be chain folding initiation sites (CFIS) according to methods developed previously. Subsequent self-organization maps (SOM) analysis has revealed key structural states involved in these early folding events.

Intrinsically disordered proteins (IDPs) contain more charged amino acids than folded proteins, resulting in a lack of hydrophobic core(s) and a tendency to adopt rapidly interconverting structures rather than well-defined structures. The structural heterogeneity of IDPs, encoded by the amino acid sequence, is closely related to their unique roles in biological pathways, which require them to interact with different binding partners. Recently, Robustelli and co-workers have demonstrated that a balanced all-atom force field can be used to sample heterogeneous structures of disordered proteins (Proc. Natl. Acad. Sci. U.S.A.2018115E4758E4766). However, such a solution requires extensive computational resources, such as Anton supercomputers. Here, we propose a simple and effective solution to sample the conformational space of IDPs using a publicly available web server, namely, the UNited-RESidue (UNRES) web server. Our proposed solution requires no investment in computational resources and no prior knowledge of UNRES. UNRES Replica Exchange Molecular Dynamics (REMD) simulations were carried out on a set of eight disordered proteins at temperatures spanning from 270 to 430 K. Utilizing the latest UNRES force field designed for structured proteins, with proper selections of temperatures, we were able to produce comparable results to all-atom force fields as reported in work done by Robustelli and co-workers. In addition, NMR observables and the radius of gyration calculated from UNRES ensembles were directly compared with the experimental data to further evaluate the accuracy of the UNRES model at all temperatures. Our results suggest that carrying out the UNRES simulations at optimal temperatures using the UNRES web server can be a good alternative to sample heterogeneous structures of IDPs.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products biosynthesized from a genetically encoded precursor peptide. The enzymes that install the post-translational modifications on these peptides have the potential to be useful catalysts in the production of natural-product-like compounds and can install non-proteogenic amino acids in peptides and proteins. However, engineering these enzymes has been somewhat limited, due in part to limited structural information on enzymes in the same families that nonetheless exhibit different substrate selectivities. Despite AlphaFold2’s superior performance in single-chain protein structure prediction, its multimer version lacks accuracy and requires high-end GPUs, which are not typically available to most research groups. Additionally, the default parameters of AlphaFold2 may not be optimal for predicting complex structures like RiPP biosynthetic enzymes, due to their dynamic binding and substrate-modifying mechanisms. This study assessed the efficacy of the structure prediction program ColabFold (a variant of AlphaFold2) in modeling RiPP biosynthetic enzymes in both monomeric and dimeric forms. After extensive benchmarking, it was found that there were no statistically significant differences in the accuracy of the predicted structures, regardless of the various possible prediction parameters that were examined, and that with the default parameters, ColabFold was able to produce accurate models. We then generated additional structural predictions for select RiPP biosynthetic enzymes from multiple protein families and biosynthetic pathways. Our findings can serve as a reference for future enzyme engineering complemented by AlphaFold-related tools.